The detection, the characterization, the identification of biological species and in particular of organisms such as bacteria is a need which relates to many fields, for example medical or veterinary diagnostic, food safety or the environment. This is also a need in various fields of production, in biotechnology and pharmaceuticals. Finally, this is a need in many fields of research in life sciences and in pharmacy.
A particularly important example is that of bacteria. If some are used in industry for the production of foodstuffs, of chemical products or drugs, but also for the treatment of wastewaters, others may prove to be dangerous for health. In many cases, a rapid detection and of identification of these bacteria is therefore critical. Moreover, it is often not sufficient to detect traces of bacteria, but it is also often also necessary to be aware of their condition (live or dead), their infectivity and their resistance to such or such an antibiotic.
Therefore, it is desirable to know how to detect bacteria in a sample possibly containing a large variety of microorganisms. The response time has to be as short as possible, the confidence as high as possible, and the handling operations to be carried out as simple as possible.
Presently, the standard system for identifying bacteria involves the culture of colonies on a more or less selective medium depending on cases, and manual counting, or analysis of the content of the colonies after culture, for example, by molecular analysis technologies like mass spectrometry. It is then necessary to wait for approximately 24 to 48 hours for the development of the bacteria. Further, the required facilities, as well as the personnel, have to be specialized, even if computer programming tools may be useful. In order to identify bacteria strains, a morphological comparison, tests of resistance to antibiotics, or an identification by a colorimetric method, or a combination of such characterizations should further be carried out. This method is therefore inexpensive, quite reliable and easy to implement, but it requires an important concentration of bacteria, as well as a very significant operation time.
In document CN 102094062, a method was proposed in which the sample is passed through a filter, so that the bacteria accumulate in the filter; these bacteria are then cultured by placing the filter in a culture medium, and the metabolites from the growth of the bacteria are detected. This method is complex and requires a long analysis time.
It is also known how to culture cells in a system based on fluidized beds. For example, in document CN 201933088 a system was proposed for the culture of mammal cells adherent on a solid substrate, comprising a fluidized bed comprised between two porous plates, and perfused by a gas for promoting the culture. This system is, however, not suitable for diagnostic or detection applications on samples of a small volume.
In document CN 1303924, a method was proposed for producing viruses inside animal cells, themselves adherent to particles maintained within a magnetically stabilized fluidized bed. The method provides oxygenation, exchange of culture medium, and the collection of viruses at the outlet of the chamber. This method is adapted for production; it requires the preliminary injection of a large number of cells and therefore does not allow the detection of rare cells.
In document WO 86/05202 still another cell culture system was proposed on a fluidized bed, for the culture of tissues or fermentation, involving the treatment of a portion of the treated fluid in the fluidized bed in an ancillary chamber, and its reinjection into the bed. This system further requires a large initial number of cells, and is therefore not adapted to the detection of rare cells.
Other studies focused on more direct and sensitive detection methods, in microfluidic devices.
For example, in the article Soft Inertial Microfluidics for High Throughput Separation of Bacteria from Human Blood Cells, in Lab Chip, 9:1193-1199 (2009), a continuous separation of cells by a physical method, by using the relationship between the inertia of the objects to be sorted and their characteristic dimensions, was described. This method makes it possible to work at a high flow rate, but the separation remains inaccurate in terms of resolution size. Moreover, many bacteria or different cells may have the same size, and this sorting criterion is therefore insufficient.
Electrophoretic sorting methods may also be used. Indeed, the electrophoretic mobility of a bacterium depends on the charge and on the radius of the bacterium. Electrophoresis thus makes it possible to concentrate the sought bacteria, but also to discard the dead bacteria, by modifying the electric direction of the applied field over very short times, as described in the article Enrichment of viable bacteria in a micro-volume by Free-flow electrophoresis, in Lab Chip, 12: 451-457(2012). Alternatively, the dielectrophoresis force which is based on the difference of dielectric constants of the objects to be sorted out, may be used.
Such methods have to be coupled with PCR (polymerization chain reaction) systems or with other genomic analyses in order to identify the bacteria which are present, as discussed in the article A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva, in Lab Chip, 13: 1325-1332 (2013). However, specificity and sensitivity often remain insufficient.
Still another method is the capture by binding between antibody and antigen. Mention may notably be made of a use of this method for capturing bacteria by directly grafting antibodies on the microfluidic channels of a circuit, in the article On-chip microfluidic biosensor for bacterial detection and identification, in Sensors and Actuators B, 126: 508-514 (2007). In this case, the grafting of the antibodies is carried out by silanization and it is necessary to let the bacteria settle so that the capture is efficient. The flow rate is insufficient when it is required to detect a few bacteria in large volume samples, as is the case for example for diagnostic analyses and food safety analyses.
In the article Immunomagnetic bead-based cell concentration microdevice for dilute pathogen detection, in Biomed Microdevices, 10:909-917 (2008), a microfluidic system is described wherein magnetic beads bearing antibodies are immobilized by a side magnet, in order to capture bacteria. However, the capture efficiency is only of 2 CFU per μL, which is very insufficient for the required efficiency levels, for example in diagnostic or food analysis applications.
In document WO 2014/037674 a microfluidic fluidized bed is described, with a circulation of magnetic beads controlled by an external magnetic field, notably for the capture of bacteria, still by grafting specific antibodies on the magnetic beads. However, this system does not have sufficient sensitivity for detecting a few bacteria in large volumes of samples. Moreover, it does not make it possible to differentiate live bacteria from dead bacteria.
There are also cell types which are particularly interesting to characterize, to concentrate, to culture and/or to amplify: for example, these are yeasts, or superior eukaryotic cells, notably mammal cells and particularly human cells. For example, biotechnology and medicine, notably regenerative medicine or graft medicine, are interested in novel methods for characterization, for sorting and for developing stem cells, progenitor cells, pluripotent or totipotent cells. For example, in the article Microfluidic Capture of Endothelial Colony-Forming Cells from Human Adult Peripheral Blood: Phenotypic and Functional Validation In Vivo, in Tissue Engineering Part C, vol. 21: 274-283 (2015), doi: 10.1089/ten.tec.2014.0323, Lin et al. describe a method allowing the microfluidic capture of human cells, endothelial colony forming cells (ECFC). However, in this method, microfluidics is only used for capture, and it is then necessary to transfer the cells of interest into conventional culture plates, which leads to a method which is long and expensive in handling operation time.
Therefore, there is an actual need for developing systems for detecting rare bacteria in quite significant volumes of samples, in an easy, automated and rapid manner, and for identifying their infectivity or their proliferation potential. Similar problems also exist for other unicellular or pluricellular organisms, like yeasts or parasites, or eukaryotic cells, for example stem cell or cancer cells.